Synthesis, DNA binding, hemolytic, and anti-cancer assays of curcumin I-based ligands and their ruthenium(III) complexes

Article abstract of DOI:10.1007/s00044-012-0133-8

Knoevenagel condensates of curcumin I were synthesized with p-hydroxybenzaldehyde and 4-hydroxy-3,5-dimethoxy benzaldehyde and allowed to react with semicarbazide to form the corresponding curcumin I-based ligands. The ligands were complexed with ruthenium(III) metal ions. These complexes (C ₁ and C₂) were purified by chromatography and characterized as octahedral geometries by analytical techniques. The binding affinities of these compounds for calf thymus DNA were determined. DNA binding constants (K _b ) for the two complexes were 1.46 × 10 ⁴ and 3.54 × 10⁴ M^-1, respectively. Similarly, the binding constants (K _sv) for C₁ and C ₂ were 9.40 × 10³ and 9.30 × 10³ M^-1, respectively. Hemolytic assays of the compounds showed less toxicity than the standard anti-cancer drug letrazole. The compounds showed good activity against the cervical cancer cell line (HeLa) and moderate activity against liver hepatocellular carcinoma (HepG2), breast cancer (MDA-MB-231) and human colon adenocarcinoma (HT-29) cells lines. These compounds showed potential for treatment of cervical cancer in the future.

Full text of DOI:10.1007/s00044-012-0133-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:1386–1398
DOI 10.1007/s00044-012-0133-8
ORIGINAL RESEARCH
Synthesis, DNA binding, hemolytic, and anti-cancer assays
of curcumin I-based ligands and their ruthenium(III) complexes
•
•
•
Imran Ali Kishwar Saleem Diana Wesselinova
Ashanul Haque
Received: 10 October 2011 / Accepted: 26 May 2012 / Published online: 17 June 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Knoevenagel condensates of curcumin I were
synthesized with p-hydroxybenzaldehyde and 4-hydroxy-
3,5-dimethoxy benzaldehyde and allowed to react with
semicarbazide to form the corresponding curcumin I-based
ligands. The ligands were complexed with ruthenium(III)
metal ions. These complexes (C1 and C2) were purified by
chromatography and characterized as octahedral geome-
tries by analytical techniques. The binding affinities of
these compounds for calf thymus DNA were determined.
DNA binding constants (Kb) for the two complexes were
1.46 9 104 and 3.54 9 104 M-1, respectively. Similarly,
the binding constants (Ksv) for C1 and C2 were 9.40 9 103
and 9.30 9 103 M-1, respectively. Hemolytic assays of the
compounds showed less toxicity than the standard anti-
cancer drug letrazole. The compounds showed good
activity against the cervical cancer cell line (HeLa) and
moderate activity against liver hepatocellular carcinoma
(HepG2), breast cancer (MDA-MB-231) and human colon
adenocarcinoma (HT-29) cells lines. These compounds
showed potential for treatment of cervical cancer in the
future.
Introduction
Many natural products have shown potential as anti-cancer
agents, i.e., some as chemotherapeutics and others in
clinical trials (Ali et al., 2010). Turmeric is a rhizome of
the Curcuma longa (Zingiberaceae) plant of which curcu-
min I [1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadi-
ene-3,5-dione] is a major constituent. In the last few
decades, curcumin has been studied extensively for its
various medicinal applications including its anti-cancer
effects (Duvoix et al., 2005; Lina and Lee, 2006; Ohtsu
et al., 2002). Currently, its efficacy is being evaluated in
phase I clinical trials for colon cancer by the National
Cancer Institute (http://www.cancer.gov/clinicaltrials/
search/view?cdrid=648267&version=HealthProfessional&
protocolsearchid=6994018). Its low molecular weight and
low toxicity have made curcumin an ideal candidate for
anti-cancer chemotherapy (Kelloff et al., 1996). However,
curcumin exhibits poor potency and absorption character-
istics, which has prompted researchers to synthesize its
derivatives (Qiu et al., 2008; Zhang et al., 2008; Ferrari
et al., 2009). The medicinal potencies of curcumin may be
enhanced by synthesizing its derivatives followed by metal
ion complexation (Anand et al., 2007; John et al., 2002).
The improved potency of curcumin derivatives may be due
to better absorption and greater kinetic stabilities when
compared to native curcumin. A number of studies have
examined the anti-cancer activity of these derivates when
complexed with metal ions. (Manoharan, 1996; Adams
et al., 2005; Aggarwal et al., 2003; Lo Tempio et al., 2005;
Parka et al., 2005). Ferrari et al. (2009) prepared some
derivatives of curcumin and tested their cytotoxicities
against 288 and C-13 cell lines. Padhye et al. (2009) also
synthesized Knoevenagel condensates and prepared their
copper complexes. These molecules were shown to inhibit
Keywords Curcumin I ꢀ Ruthenium complexes ꢀ
Chromatographic and spectroscopic studies ꢀ
DNA bindings ꢀ Hemolytic assay ꢀ Anti-cancer profile
I. Ali (&) ꢀ K. Saleem ꢀ A. Haque
Department of Chemistry, Jamia Millia Islamia (Central
University), New Delhi 110025, India
e-mail: drimran_ali@yahoo.com; drimran.chiral@gmail.com
URL: http://old.jmi.ac.in/2000/Fnat/imran_ch.htm
D. Wesselinova
Institute of Parasitology and Experimental Pathology, Bulgarian
Academy of Science, 1113 Sofia, Bulgaria
123

Med Chem Res (2013) 22:1386–1398
1387
Tumor necrosis factor-a (TNF-a)-induced nuclear factor
kappa-light-chain-enhancer of activated B cells (NF-jB)
activation and proliferation of human leukemic myeloge-
nous leukemia cell line (KBM-5) cells.
Experimental
Materials and methods
Chemicals and reagents
Ruthenium complexes demonstrate improved anti-
tumor activity when compared to other metal complexes,
including platinum-based drugs. This is due to a number
of factors including their low toxicity, potential for non-
cross-resistances and good potency (Zeller et al., 1991;
Coluccia et al., 1993). In addition, the rate of ligand
exchange, range of accessible oxidation states and an
ability to mimic iron (in binding to certain biological
molecules) make ruthenium compounds good alternatives
to other metal complexes in the treatment of cancer. The
binding capacities of ruthenium compounds with
deoxyribose nucleic acid (DNA) are also faster and
stronger. This is due to the octahedral structures of
ruthenium complexes in comparison to the square planar
geometries of platinum complexes. It is possible that
square planar geometry creates a hindrance to DNA
binding whereas octahedral complexes do not (Brabec
and Novakova, 2006). Generally, ruthenium complexes
are more stable due to four co-ordinate and two elec-
trovalent bonds which have labile entities such as chlo-
ride ions or water molecules. Therefore, metal complexes
are not taken up by the tissues while in the systemic
circulation. The whole complex remains unchanged and
targets only cancerous cells. Consequently, host toxicity
induced by metal ions is significantly reduced. As soon
as the metal complex reaches hypoxic area, the metal
ions gets reduced in situ; leading to ruthenium accu-
mulation in tumor masses (Mestroni et al., 1993; Frasca
et al., 1996).
The rhizome of C. longa was collected from the agricul-
tural field, New Delhi, India. The plant was identified
by observing its taxonomical features of the plant.
p-hydroxybenzaldehyde, 4-hydroxy-3,5-dimethoxybenzal-
dehyde, semicarbazide, tris-(hydroxymethyl)aminometh-
ane were obtained from Sisco Research Lab., Mumbai,
India and S.D. Fine Chem. Ltd., New Delhi, India. Ethanol,
methanol, chloroform, dichloromethane and hexane of
HPLC grade were purchased from Merck, Mumbai, India.
CT-DNA (as sodium salt) was obtained from SRL Pvt. Ltd.
Mumbai, India. The concentrations of the DNA were
determined spectrophotometrically with an extinction
coefficient of 6,600 M-1 cm-1 at 260 nm. Silica gel G
(10–40 lm) for thin layer chromatography (TLC) and
normal silica gel (60–120 lm) for column chromatography
were supplied by Merck, Mumbai, India. Ruthenium tri-
chloride dihydrate of A.R. grade was obtained from Ava-
rice Lab. Pvt. Ltd., G.B. Nagar, India. Tris–HCl Buffer
(1.0 9 10-2 M) was prepared in Millipore water at pH
range of 7.2–7.4.
Instruments used
The percentages of C, H, and N were determined by using
Vario EL elemental analyzer. Molar conductance mea-
surements were carried out on Decibel conductivity meter at
room temperature. UV–Vis spectra were obtained by using
T80 UV–Vis spectrophotometer. FT-IR spectra were
obtained in the range of 4,000–400 cm-1 on a Nicolet
(Nexus 670) Fourier transform infrared (FT-IR) spectrom-
eter. Nuclear magnetic resonance (1H and 13C NMR)
spectra were recorded by using Bruker 300 MHz instru-
ment. Ultraviolet (UV) cabinet was used to view TLC
(2.5 cm 9 7.5 cm) plates. pH meter of Control Dynamics
(Model APX 175 E/C) was used to record pH of the solu-
tions. Emission spectra of the complexes were recorded on
Jobin-Yvon Fluorolog 3-22 Spectrofluorimeter; using
450 W xenon lamp and R928P PMT as the excitation
source and detector, respectively. The melting points were
determined on Veego instrument (Model REC2203882).
Millipore water was prepared by using a Millipore Milli-Q
(Bedford, MA USA) water purification system. HPLC
system was from ECOM (Czech Republic) consisting
of solvent delivery pump (model Alpha 10), manual
injector, absorbance detector (Sapphire 600 UV–Vis),
Two new curcumin I condensates (ligands) were syn-
thesized by replacing the active hydrogen of curcumin with
p-hydroxybenzaldehyde and 4-hydroxy-3,5-dimethoxy-
benzaldehyde, respectively (Knoevenagel condensation).
These condensates of curcumin I were synthesized using
piperidine as a catalyst. Ruthenium metal complexes of
these condensates were then prepared. These complexes
were purified by chromatography and characterized by
different analytical techniques including elemental analy-
sis, molar conductivity, UV, FT-IR 1H, 13C NMR, and
mass spectroscopy. The interaction of the prepared com-
plexes with calf thymus DNA (CT-DNA) was studied at
physiological pH. The cytotoxic effects of the ligands and
complexes on human RBCs were studied. Finally, the
activity of these molecules on liver hepatocellular carci-
noma (HepG2), breast cancer (MDA-MB-231), cervical
cancer (HeLa) and human colon adenocarcinoma (HT-29)
cell lines was determined.
123

1388
Med Chem Res (2013) 22:1386–1398
Synthesis of complexes
Chromatography I/F module data integrator (Indtech.
Instrument, India) and Winchrome software was used to
determine the purity of curcumin based ligands and their
metal complexes. The column used was Sunniest C18
(150 9 4.5 mm, 5.0 lm) Chromanik, Japan. For cell line
activities, absorbance of each well was read at 490 nm by
an automatic microplate reader (‘‘Tecan’’, Austria).
Complexes C1 and C2 were synthesized from ligands L1
and L2, separately and respectively. The solution of
ruthenium salt (26.14 mg, 0.1 mM, in 3–5 mL methanol)
was added dropwise to L1 solution (58.6 mg, 0.1 mM, in
20.0 mL methanol). The reaction mixture was stirred for
3 h at room temperature. The concentration was adjusted to
5.0 mL. A solid complex C1 was obtained which was fil-
tered and dried. The product was washed with a small
amount of ether and hexane, separately, respectively. The
purity of the complex was ascertained by HPLC using
water-acetonitrile (80:20, v/v) as mobile phase. The same
procedure was applied for the synthesis of C2.
Separation of curcumin
First of all, the rhizome was ground to powder. The cur-
cumin mixture was extracted from the powder using eth-
anol. The curcumin mixture was then separated by column
chromatography. A glass column (35 cm 9 2.5 cm) was
packed with 10 % NaHCO3 impregnated silica gel and
eluted using pure dichloromethane. The separation and
purity of curcumin were determined by TLC with dichlo-
romethane–methanol (DCM–MeOH) (99:1, v/v) after
visualization in a UV cabinet.
Characterization of ligands and metal complexes
The compositions of P1, P2, L1, L2, C1, and C2 were
determined by the elemental analysis. Furthermore, molar
conductance was also used to determine the ratio and
nature of metal ion complexes. The spectroscopic studies
Ligand synthesis
such as UV–Vis, FT-IR, and H & 13C NMR were also
1
used to characterize the synthesized compounds. The per-
centage values of various elements in these compounds
were also calculated by Chem. Draw software (theoretical
values). The observed and theoretical values were com-
pared with each other.
Curcumin I (0.2 mM, 73.6 mg) was dissolved in a mixture
of chloroform/methanol (*15 mL) in a round bottom
flask. A methanolic solution of p-hydroxybenzaldehyde
(0.2 mM, 24.4 mg) was added while stirring continuously.
A small amount of catalyst (piperidine, *2–5 %v/v) was
added to the mixture which was then stirred at room tem-
perature for 48 h. The progress of the reaction was moni-
tored by TLC [DCM–MeOH (99:1, v/v)]. After completion
of the reaction, the liquid was poured into a solution
(*30 mL) of hexane–chloroform (50:50, v/v) mixture,
which resulted in a light brown product (P1), [4-(4-hydroxy
benzyl)-1,7-bis(4-hydroxy,3-methoxyphenyl)-1,6-heptadi-
ene-3,5-dione]. The product was separated and washed
with petroleum ether to remove un-reacted reagents. The
Schiff base of the above product was synthesized by dis-
solving P1 (0.3 mM, 141.6 mg) into methanol (25 mL)
and stirring in the presence of semicarbazide hydrochlo-
ride (0.6 mM, 66.9 mg, with some drops of water) at
room temperature for 24 h. A brown solid (ligand; L1)
[4-(4-hydroxybenzyl)-1,7-bis(4-hydroxy,3-methoxyphenyl)-1,
6-heptadiene-3,5-disemicarbazone] was obtained, which was
washed with petroleum ether followed by its recrystallization
using chloroform-hexane mixture (70:30, v/v). The same
procedure was applied for the synthesis of second ligand
L2 [(4-(4-hydroxy-3,5-dimethoxy benzyl)-1,7-bis(4-hydroxy,
3-methoxyphenyl)-1,6-heptadiene-3,5-disemicarbazone] with
4-hydroxy-3,5-dimethoxybenzaldehyde through P2, [4-(4-
hydroxy-3,5-dimethoxybenzyl)-1,7-bis(4-hydroxy,3-methoxy-
phenyl)-1,6-heptadiene-3,5-dione] species.
Interactions of metal complexes with DNA
To study the biological activities of the synthesized metal
complexes, binding experiments were carried out with CT-
DNA spectroscopically. UV–Vis and fluorometry spectro-
scopic techniques were used. The experimental protocols
of these techniques are outlined below.
UV spectroscopy
The different concentrations of DNA used were
0.4 9 10-4 M to 1.0 9 10-4 M for C1 (1.0 9 10-4 M)
and 1.8 9 10-4 to 2.4 9 10-4 for C2 (1.8 9 10-4 M).
First of all kmax of pure DNA and metal complexes were
recorded along with their absorbance’s. The purity of DNA
was determined by taking ratio of A260/A280 (A260
/
A280 C 1.80 indicating that DNA was sufficiently free of
protein). The concentration of stock solution of DNA was
determined by using e = 6,600 M-1 cm-1 at 260 nm.
1.5 mL of each solution of DNA and metal complex was
mixed together followed by recording kmax and absorbance.
The absorption spectra were recorded after each addition of
different concentration of DNA solution (1.5 mL).
123

Med Chem Res (2013) 22:1386–1398
1389
Fluorometric spectroscopy
concentration of 0.5 mg/mL. The investigation was carried
out by dilution of stock solution in the ratio of 1:10, 1:100,
1:1,000, and 1:10,000. The samples of cells, grown in
non-modified medium, served as controls. After 24 h of
incubation, MTS colorimetric assay of cell survival was
performed. The wells were treated with MTS solution and
incubated for 2 h at 378 C under 5 % carbon dioxide and
95 % air atmosphere. The absorbance of each well at
490 nm was read by an automatic microplate reader
(‘‘Tecan’’, Austria). Relative cell viability, expressed as a
percentage of the untreated control (100 % viability), was
calculated for each concentration. The percentage inhibi-
tions of the reported compounds were calculated by origin
program.
Luminescence quenching experiments were conducted by
adding aliquots of varying concentrations of [Fe(CN)6]4-
to the metal complexes (6.1 9 10-6 M) both in the absence
and presence of CT-DNA (6.1 9 10-4 M) in Tris–HCl
buffer. The experiment was carried out in 10-2 M Tris
buffer; with pH 7.2–7.4 adjusted by dilute HCl.
Cytotoxicity assays
Hemolytic profiles
The hemolytic activities of the synthesized compounds
were determined on human red blood cells (RBCs). Human
blood from healthy individuals was collected in tubes
containing EDTA as an anti-coagulant. The erythrocytes
were harvested by centrifugation for 10 min at 2,000 rpm
at 20 °C, followed by washing three times with Phosphate
buffered saline (PBS, pH 7.4, 0.0085 g/mL). PBS was
added to the pellet, to yield a 10 % (v/v) erythrocytes/PBS
suspension. The 10 % suspension was then diluted to 1:10
in PBS. From each suspension, 100 lL was added to
100 lL of different dilution series of synthesized com-
pounds along with letrazole in the same buffer in eppendorf
tubes (in triplicate). Total hemolysis was achieved with
1 % Triton X-100. The tubes were incubated for 1 h at
37 °C and then centrifuged for 10 min at 2,000 rpm at
20 °C. From the supernatant fluid, 150 lL was transferred
to a flat-bottomed microtiter plate (BIO-RAD, iMark, US),
and the absorbance was measured spectrophotometrically
at 450 nm. The hemolysis percentage was calculated by
following equation:
Result and discussion
Purification of the products
The products (P1, P2, L1, L2, C1, and C2) obtained from the
above reactions were purified by washing with a mixture of
DCM–MeOH (99:1, v/v) to remove un-reacted reagents
and other impurities. The purities of these products were
verified by TLC, HPLC, UV–Vis spectroscopy and melting
points. The sharp melting points of L1 and L2 were 102 and
116 °C, respectively. All these observations indicated quite
good purities of these products.
Characterization of the products
The prepared products were characterized by the following
methods.
ꢀ
ꢁ
ðA450 of test compound treated sample ꢁ A450 of buffer treated sampleÞ
ðA450 of 1 % Triton X ꢁ 100 treated sample ꢁ A450 of buffer treated sampleÞ
% Hemolysis ¼
ꢂ 100%
Cell line profiles
UV–Vis spectra
The cytotoxic activity of the compounds L1, L2, C1, and C2
was evaluated for four human cell lines—colorectal cancer
(HT-29), breast cancer (MDA-MB-231), human liver car-
cinoma (HepG2), and cervical cancer (HeLa) cancer using
the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeth-
oxyphenyl)-2-(4-sulfophenyl)-2H-tetrasolium inner salt]
test (Cell Titer 96 Non-Radioactive Cell Proliferation
Assay, 1999). The compounds were dissolved in DMSO at
The spectra of P1, P2, L1, L2, C1, and C2 were scanned from
200 to 800 nm. The kmax observed were at 210, 212, 224,
215, 215, and 219 nm, respectively. The red shift was
observed in the case of P1, P2, L1, and L2 species, i.e.,
210–224 in P1 to L1 and 212–215 in P2 to L2; indicating the
formation of C=N in both ligands (L1 and L2). Further-
more, the appearance of these kmax indicated the presence
of conjugated carbon–carbon double bonds. Higher energy
123

1390
Med Chem Res (2013) 22:1386–1398
Table 1 Elemental analyses of the ligands and their metal complexes
Ligands
Percentage/elements/values
C
H
N
O
Cl
L1
L2
C1
C2
61.0 (61.04)
59.1 (59.44)
45.1 (45.36)
44.6 (44.88)
4.8 (5.0)
4.6 (5.26)
3.6 (3.7)
4.4 (4.20)
14.1 (14.3)
12.6 (13.0)
10.3 (10.5)
9.5 (9.81)
19.9 (20.1)
–
21.5 (21.6)
14.0 (14.11)
16.7 (16.83)
–
13.2 (13.4)
12.1 (12.44)
The values outside and in parenthesis are experimental and theoretical
absorption bands in C1 and C2 were observed at 336.0 and
335.0 nm, respectively, which might be due to the p ? p*
transitions. Lower energy absorption bands in both com-
plexes were observed due to metal to ligand charge transfer
(MLCT) transitions. MLCTs of C1 and C2 appeared at
466.5 and 446.0 nm, respectively.
*3500, 1659, 1020–1040, *1480, 2952–2850, and 1350–
1050, which corresponded to the stretching frequencies of
–OH, –C=O, Ar–O–C, –C=C–, =C–H, and –C–H groups,
respectively. FT-IR peak values of L1 were similar to those
of P1 except –C=O stretching frequency; with additional –
C=N– and[N–H stretching frequencies at 1629 and 3573–
3590 cm-1. These results indicated the substitution reac-
tion of semicarbazide with P1 at the –C=O group. In the
ruthenium metal ion complex of L1, imine stretching fre-
quency underwent a red shift indicating co-ordination of
the Schiff base, L1, through the azomethine nitrogen atom.
This is further confirmed by the appearance of moderate
absorptions band in C1 at 510 cm-1 due to Ru–N stretching
Elemental analysis
The compositions of L1, L2, C1, and C2 were determined by
elemental analyses and the values are given in Table 1. The
theoretical percentages of the elements were also calcu-
lated and are given in Table 1, which clearly indicate a
good agreement with those of the experimental values. On
the basis of the data of elemental analyses the molecular
formulae of L1, L2, C1, and C2 were determined as
C30H30N6O7, C32H36N6O9, RuC30H30N6O7Cl3, and
RuC32H36N6O9Cl3, respectively. These results confirmed
that complex formation had occurred in a 1:1 ratio of
ligand and ruthenium metal ion.
1
vibrations (Priya et al., 2009). H NMR d values observed
for P1 were 3.46, 6.66, 6.81, 6.83, 7.52, 7.32, and 7.57
indicating the presence of protons of –OCH3(6H),
ArH(2H), ArH(4H), =C–H (2H), H–C=(2H), ArH(4H), and
=CH–Ar(1H) groups, respectively. 1H NMR d values of L1
were almost the same as those of P1 except for additional
values of 2.50 and 6.78, which corresponded to –NH2 (4H)
and [NH (2H) groups, respectively. The various d values
of 13C NMR for P1 species in (DMSO-d6) were: 197.47,
(–C=O), 167.43 (–C=C–), 153.930 (Ar–C=), 148.19
(methylene), 123.88 (=C–C=O), 57.45 (–OCH3), (160,
143.46, 142.51, 129.18, 125.57, 121.86, 120.56, 119.68,
115.63, 115.43, aromatic). Furthermore, ESI–MS (m/z):
[M?] 472, confirmed the structure of P1 as shown in Fig. 1.
The various groups identified in L1 by 13C NMR were:
164.65 (-CONH2), 161.74 (–C=C–), 156.30 (–C=N),
123.74 (=C–C=N–), 147.45 (methylene), 57.60 (OCH3),
(159.87, 143.40, 141.45, 129.28, 125.58, 121.86, 120.45,
119.62, 115.62, 115.53, Aromatic). ESI–MS (m/z) value:
[M ? H]? 587 supported the structure of L1, while ESI–
MS (m/z) value of C1: [M ? Na]? 817 was in good
agreement with the proposed structure.
Conductance measurement
The molar conductivities of C1 and C2 complexes were
found in the range of 104–110 X-1 cm2 M-1 suggesting 1:1
ionic nature of the complexes (Chittilapilly and Yusuff
2008). Thus, it can be concluded that two Cl-1 anions exist
within the co-ordination sphere of complexes (C1 and C2),
forming two electrovalent bonds with ruthenium metal ion.
One of the chloride anions exists outside the co-ordination
sphere of the complexes to balance the excess one unit
charge on the metal ions. Therefore, the molecular formulae
of the complexes C1 and C2 may be written as [RuC30H30-
N6O7Cl2]Cl and [RuC32H36N6O9Cl2]Cl, respectively.
1
FT-IR and H and 13C NMR and MS characterization
Similarly, the peaks of P2 in FT-IR were observed
almost in the same region as that of P1, i.e., *3500, 1650,
1040, *1470, 2950–2850, and 1400–1083, which corre-
sponded to the stretching frequencies of –OH, C=O, Ar–O–
C, C=C, =C–H, and C–H groups, respectively. In the case
The structures of the synthesized species were confirmed
by recording their FT-IR, NMR and MS spectra. The
various peaks of P1 obtained in FT-IR spectra were at
123

Med Chem Res (2013) 22:1386–1398
1391
Fig. 1 Models of a C1 and b C2 ruthenium metal ion complexes. Asterisk nitrogen atoms (H atom have been omitted)
of L2, the carbonyl stretching frequency was replaced by
C=N and[N–H stretching frequencies at 1,630 and 3,580–
3,590 cm-1, respectively. For C2, the frequency due to the
azomethine group also underwent a red shift with Ru–N
compounds calculated from chemdraw model were in good
agreement with those of the experimental ones.
Structures of ruthenium metal ion complexes
1
stretching vibrations at 490 cm-1. The H NMR d values
observed for P2 were 3.79, 6.66, 6.72, 7.16, 7.57, 7.52, and
8.35 indicating the presence of protons of –OCH3(6H),
ArH(2H), ArH(4H), =C–H (2H), H–C=(2H), ArH(4H), and
Based on the findings outlined above, the structures of C1
and C2 were drawn with the help of Pymol software and are
shown in Fig. 1. These models clearly show that both L1
and ligands formed co-ordinate bonds with ruthenium
metal ions through azomethine nitrogen and oxygen atoms,
respectively. Figure 1 also depicts that two chloride anions
formed electrovalent bonds. This arrangement of ligands,
chloride anions and ruthenium metal ions results in the
octahedral geometries of the reported complexes.
1
=CH–Ar(1H) groups, respectively. H NMR spectra of L2
contained additional protons of –NH2 (4H) and[NH (2 H)
groups at 2.69 and 6.62, d values, respectively The dif-
ferent FT-IR and H NMR values of P1, P2, L1, and L2 are
1
given in Tables 2 and 3, respectively. Based on the
experimental knowledge and characterization, the synthe-
ses protocols of these products were developed and given
in Schemes 1 and 2, respectively. Finally, the developed
structures of P1, P2, L1, L2, C1, and C2 were verified by
drawing their structures with the help of chemdraw and
Pymol software. FT-IR and NMR values of these
DNA-binding studies
As discussed above, the interactions of the synthesized
metal complexes with CT-DNA were investigated by using
Table 2 FT-IR spectral values of Knoevenagel condensates and its Schiff bases (cm-1
)
Knoeven condensates and
its Schiff bases
mOH br
mC=O
mC=N
mN–H str.
mAr–O–C
mC=C
m =
mC–H
C–H, C–H
P1
L1
P2
L2
*,3500
*3,500
*3,500
*3,500
1,659
Absent
1,629
Absent
1,020–1,040
1,038
*1,480
*1,480
1,470
2,952, 2,850
2,950, 2,840
*2950–2,850
2,952, b2847
1,350, 1,050
Absent
1650
3,573–3,590
Absent
1,350, 1,100
*1,400, 1,083
1,380, 1,126
Absent
*1630
1,040
Absent
3,580, 3,590
1,038
1,477
–NH2 stretching frequencies of C1 and C2 was difficult to assign as both –NH2 and
–OH appears in the same region (Arjmand and Jamsheera, 2011)
Table 3 1H NMR spectral values of Knoevenagel condensates and its Schiff bases (in d Hz, DMSO)
Compounds OCH3 (6H) ArH (2H) ArH (4H) =C–H (2H) H–C=(2H) ArH (4H) =CH–Ar (1H) –NH2 (4H) –NH (2H)
P1
L1
P2
L2
3.46
3.75
3.79
3.80
6.66
6.73
6.66
6.72
6.81
6.84
6.72
6.84
6.83
7.19
7.16
7.13
7.52
7.57
7.57
7.51
7.32
7.51
7.52
7.45
7.57
8.32
8.35
8.33
Absent
2.50
Absent
6.78
Absent
2.69
Absent
6.62
123

1392
Med Chem Res (2013) 22:1386–1398
O
H
O
O
O
O
OMe
OH
MeO
Piperidine, stirring
Room temperature
OMe
OH
MeO
HO
+
CH
HO
OH
p-hydroxy benzaldehyde
(1E, 6E)-1,7 bis(4-hydroxy-3-methoxyphenyl)
hepta-1,6 diene-3,5-dione
OH
P1
O
O
OMe
OH
O
O
N
NH2
NH
MeO
HO
Piperidine, Stirring
Room Temperature
H2N
O
+
CH
HN
H2N
. HCl
NH2
N
N
H
OMe
MeO
CH
Semicarbazide
hydrochloride
OH
HO
OH
P1
OH
L1
+
Cl
H2N
OH
N
NH2
HO
O
N
O
N
NH2
NH
H2N
Ru
Cl
N
N
HN
N
OMe
MeO
OMe
MeO
Stirring, 25 °C
Cl
+
RuCl3
CH
CH
OH
HO
OH
HO
Ruthenium Trichloride
OH
L1
OH
C1
Scheme 1 Syntheses of ligand (L1) and its ruthenium metal ion complex
UV–Vis and fluorometric spectroscopic techniques. The
results of these interactions are discussed in the following
sub-sections.
complexes, which constitutes concrete proof of DNA bind-
ing with ruthenium metal ion complexes. To calculate metal
complex–DNA binding constant (Kb), the data were treated
according to the following equations:
DNA þ Complex $ DNAꢁComplex Adduct
Kb ¼ DNAꢁComplex Adduct= ½DNAꢃ uncomplexed
ꢂ ½Complexꢃ complexed
UV spectroscopy
DNA binding studies are important for the rational design
and development of new and more efficient drugs. The
spectral variations reflect the changes in DNA conformation.
The structure and the extent of spectral changes are related to
the strength of binding (Xu et al., 2002); (Wang and Yan
2005). Therefore, anti-cancer activities of the synthesized
complexes were assessed by determining their binding
affinities with CT-DNA as described in the experimental
section. The spectra of DNA binding with C1 and C2 metal
complexes are given in Figs. 2 and 3, respectively. The
reported spectra show immediate DNA mixing with metal
complexes; confirming the fast reaction of DNA with these
complexes. Moreover, the hypochromic and bathochromic
effects were observed on interaction of DNA with both
The values of the binding constants (Kb) were obtained
from DNA absorption at 260 nm as per the standard
methods (Stephanos et al., 1996; Andrushchenko et al.,
2002). For binding affinities, the data were treated using
linear reciprocal plots based on the following equation:
1
1
1
1
¼
þ
ꢂ
A ꢁ A0
A
1 ꢁ A0 K ðA1 ꢁ A0Þ ½Ligandꢃ
where A0, A?, and A are the absorbance’s of DNA at
260 nm in the absence of ligand, metal complex–DNA
adduct and different DNA concentrations. The plots of
1/(A - A0) versus 1/[DNA] were linear. The binding
123

Med Chem Res (2013) 22:1386–1398
1393
Fig. 3 Absorption spectra of C2 complex with increasing concentra-
tions of CT-DNA (arrow decrease of the intensity on increasing DNA
concentration). Kb was calculated by ratio of intercept and slope of
plot between 1/(A0 - A) and 1/[DNA]. The details are given in
experimental and ‘‘Results and discussion’’ sections. Inset plot of 1/
A0 - A versus 1/[DNA] for complex 2
Fig. 2 Absorption spectra of C1 complex with increasing concentra-
tions of CT-DNA (arrow the decrease of the intensity on increasing
DNA concentration). Kb was calculated by ratio of intercept and slope
of plot between 1/(A0 - A) and 1/[DNA]. The details are given in
experimental and ‘‘Results and discussion’’ sections. Inset: Plot of
1/A0-A vs 1/[DNA] for complex 1
was assumed that ruthenium cations caused hypochromism
via DNA binding through phosphate groups leading to
contraction in the DNA helix axis. Furthermore, strong
hypochromic effects (with moderate bathochromic shift of
2–3 and 3–5 nm for C1 and C2, respectively) were indi-
cations of their good DNA binding propensities (Liu et al.,
2003). These results illustrated that the mode of binding of
complexes was quite different from other metal based ion
anti-cancer drugs such as cis-platin (cross-linking through
DNA bases) and titanocene dichloride (binding through the
phosphate group of DNA only). Therefore, the developed
and reported ruthenium metal ion complexes have different
binding modalities, which may be more beneficial in anti-
cancer chemotherapy.
constants (Kb) were obtained from the ratio of the inter-
cepts and the slopes of the plots between 1(A - A0) and
1/[DNA] (Figs. 2, 3). The values for Kb were found to be
1.46 9 104 and 3.54 9 104 M-1 for C1 and C2, respec-
tively. Hyper- and hypochromicities were indicative of
changes in double helical structure of DNA. Hyperchromic
and hypochromic effects were responsible for the changes
in DNA conformation and structure after DNA–complex
adduct formation. The hypochromism was due to the
contraction of DNA helix axis, while hyperchromism
resulted from the damage of DNA double helix structure.
The hypochromic shift was attributed to the intercalative
binding mode of both complexes with DNA. The spectral
hypochromic effect might be due to the presence of
enhanced positive charge of cations, which bonded to DNA
via non-covalent interactions. Presumably, it was due to
strong electrostatic attractions to the phosphate group of
the DNA backbone; leading to contraction and overall
damage to the secondary structure of DNA (Li et al., 1996;
Barton et al., 1984). The interaction behaviors of L1 and L2
with DNA were similar to C1 and C2 compounds. There-
fore, the results of L1 and L2 are not discussed herein. It
Fluorometric spectroscopy
Fluorescence spectroscopy was used to characterize the
binding characteristics of the chromophore with other mol-
ecules (Modukuru et al., 2005). At room temperature, C1
exhibited strong emission bands around 432 nm with a
shoulder at 388 nm when excited at 339 nm. For C2 an
excitation wavelength of 338 nm was used and total emis-
sion intensity monitored at 440 nm. Upon addition of
increasing amounts of CT-DNA (6.1– 6.7 9 10-6 M) to the
fixed amount of complex concentration (6.1 9 10-6 M) the
123

1394
Med Chem Res (2013) 22:1386–1398
Fig. 6 Emission quenching curves of C1 and C2. In absence of DNA;
C1 (filled square) and C2 (filled inverted triangle). In presence of
DNA; C1 (filled circle) and C2 (open star). With increasing
concentration of quencher Fe(CN)6]4- [complex] = 6.1 9 10-6 M,
Fig. 4 Emission spectra of C1 in Tris–HCl buffer containing DNA.
[DNA] = 6.1 9 10-6– 6.7 9 10-6 M. Arrow shows the intensity
changes upon increasing concentration of the DNA. Inset plot of
relative emission intensity vs [DNA]/[complex]
[DNA] = 6.1 9 10-4
M
fluorescence intensity as a function of CT-DNA concentra-
tion (in terms of [DNA]/[M]) was plotted in the insets of
Figs. 4 and 5. There was a large reduction in the emission
intensity measured for C2 in the presence of DNA, in com-
parison to C1. Generally, oxidative damage to DNA bases
occurred at guanine, which has the lowest oxidation potential
of all the DNA bases (Erkkila et al., 1999). Therefore, the
developed drugs would be expected to interact with the
damaged DNA guanine base resulting in energy/electron
transfer from the guanine base of DNA to the MLCT of the
complexes. To further demonstrate the interaction patterns
of C1 and C2 with DNA, [Fe(CN)6]4- quenching experi-
ments were performed. [Fe(CN)6]4- is a dynamic fluores-
cence quencher providing sensitive tool to examine the
nature of the interaction of the probe with the DNA (Mido-
rikawa and Kawanishi 2001). The quenching efficiency was
evaluated by the Stern–Volmer constant Ksv, which varied
with the experimental conditions:
I0=I ¼ 1 þ Ksvr
where I0 and I are the emission intensities in the absence
and the presence of the complex, respectively. Ksv is the
linear Stern–Volmer quenching constant with r as the ratio
of total concentration of complex to that of CT-DNA,
[complex]/[DNA]. A large value of binding constant
reflects weaker binding of complexes with DNA and vice
versa. Figure 6 shows the Stern–Volmer plots with ferro-
cyanide anion as quencher for C1 and C2. The plots of free
C1 and C2 complexes gave Ksv as 9.4 9 103 and
Fig. 5 Emission spectra of C2 in Tris–HCl buffer containing DNA.
[DNA] = 6.1 9 10-6– 6.7 9 10-6 M. Arrow intensity changes upon
increasing concentration of the [DNA]. Inset plot of relative emission
intensity versus [DNA]/[complex]
emission intensity of C1 and C2 decreased appreciably as
shown in Figs. 4 and 5, respectively. Titration of CT-DNA
led to a remarkable decrease in the emission intensity of C1
and C2, as illustrated in these figures. The relative
123

Med Chem Res (2013) 22:1386–1398
1395
Table 4 The percent inhibition of the reported compounds on dif-
ferent cell lines
Cell lines
Compounds:
HeLa
Concentrations
(mg/mL)
Percent inhibition
L1 C1
L2
C2
0.5
13.82 28.57 53.15 N.D
35 22.26 28.06 22.39
0.05
0.005
0.0005
0.5
33.48 20.69 15.84 16.39
30.79 24.48 40.11 39.55
MDA-MB-
231
ND
ND
ND
33.64 ND
0.05
7.64
ND
ND
8.79
ND
0.005
0.0005
0.5
12.95 ND
21.69 ND
ND
11.37
HT-29
HepG2
ND
ND
ND
ND
0.74
ND
0.51
ND
ND
ND
ND
ND
ND
ND
ND
ND
46.35 ND
0.05
ND
ND
ND
6.59
ND
ND
0.005
0.0005
0.5
48.08 ND
0.05
ND
ND
ND
ND
ND
ND
0.005
0.0005
Fig. 7 Hemolysis caused by L1, L2, C1, C2, and Letrazole Standard
anticancer drug). Hemolysis was determined by an absorbance
reading at 450 nm and compared to hemolysis achieved with 1 %
Triton X-100 (reference for 100 % hemolysis
ND not defined
L2, C1, C2 and the reference compound letrazole showed
hemolysis of 8, 2, 11, 18, and 22 % at a concentration of
5.0 lg/mL. At 10.0 and 20.0 lg/mL concentrations, the
values of hemolysis were 10, 4, 17, 28 & 32 % and 12, 3.9,
20, 29, and 60 %, respectively, for the same species. At
higher concentrations (50.0 and 100.0 lg/mL), the per-
centage hemolysis was increased to 18, 9, 24, 33 and 80 %
and 21, 15, 28, 48, and 98 %, respectively. The compara-
tive study of synthesized compounds with the conventional
anti-cancer drug indicates that the synthesized compounds
were significantly less cytotoxic at lower concentrations.
9.3 9 103 M-1, respectively. In the presence of DNA, the
quenching curve was markedly depressed, indicating that
the complexes were interacting with DNA helix. The
greater decrease in Ksv value of C2; in the presence of DNA
as compared to C1; indicated that C2 was bound more
tightly to DNA. These results indicate stronger binding of
C2 than C1, which might be due to different extents of
interior and exterior interactions of C1 and C2 with DNA.
The C2 molecule may have a stronger electrostatic inter-
action with DNA than C1. It may be due to the fact that
complex free in solution or bound on to the helix exterior is
accessible to the quencher, while another one bound to the
helix interior is much less accessible. This explanation is
also supported by the earlier works of Wu et al. (2005).
Cell line profiles
The anti-cancer profiles of the synthesized compounds
were studied on four cell lines by calculating IC50 values
and % inhibitions. The % inhibitions of the reported
compounds are given in Table 4. A perusal of this table
indicates that all four compounds are moderately active.
However, the best results have been reported on cervical
cancer (HeLa) cell line. The compounds L1 and C1 were
active for all cell lines except HT-29. The compounds L1
and C1 were active at all concentrations for the HeLa cell
line, while showing very low or no activity for other cell
lines. Compound L2 was active against HeLa cell lines at
all concentrations. It had moderate activity towards other
cell lines at higher concentrations. C2 showed activity
against HeLa cells with low or no activity on other cell
lines. The different percentage inhibitions of these
Cytotoxicity assays
Hemolytic profiles
In vitro hemolytic assays of newly synthesized compounds
are a possible screening tool for gauging in vivo toxicity to
host cells (Situ and Bobek, 2000). Human RBCs provide a
handy tool for toxicity studies of the new compounds as
they are readily available and their membrane properties
are well described. In addition, lysis may be monitored
easily by measuring the release of hemoglobin (Christie
et al., 2007). Effects of the reported compounds on human
RBCs are shown in Fig. 7. The synthesized compounds L1,
123

1396
Med Chem Res (2013) 22:1386–1398
O
H
O
O
O
O
OMe
OH
MeO
Piperidine, Stirring
Room temperature
OMe
OH
MeO
HO
+
CH
HO
MeO
OMe
OH
(1E, 6E)-1,7 bis(4-hydroxy-3-methoxyphenyl)
hepta-1,6 diene-3,5-dione
4-hydroxy-3,5-dimethoxybenzaldehyde
MeO
OMe
OH
P2
O
O
OMe
OH
O
O
N
NH2
NH
MeO
HO
Piperidine, Stirring
Room temperature
H2N
O
+
CH
HN
H2N
. HCl
NH2
N
N
H
OMe
MeO
MeO
Semicarbazide
hydrochloride
CH
OMe
OH
HO
OH
P2
MeO
OMe
OH
L2
Cl
OH
+
HO
H2N
NH2
O
N
O
NH2
NH
H2N
Ru
Cl
N
N
HN
N
N
N
OMe
MeO
OMe
OH
MeO
stirring, 25 °C
+
Cl
RuCl3
CH
CH
OH
HO
HO
Ruthenium Trichloride
MeO
OMe
MeO
OMe
OH
L2
OH
C2
Scheme 2 Syntheses of ligand (L2) and its ruthenium metal ion complex
compounds on various cell lines were due to high sensi-
tivities of the cells to the developed drugs. For the cell lines
HT-29, the maximum inhibition of 46.35 % (IC50 = ND)
at 0.5 mg/mL was shown by compound L2. For HepG-2
cell line, the maximum inhibition of 48.08 % at 0.5 mg/mL
(IC50 = ND) was observed with the compound L2. In the
case of MDA-MB-231 cell line, maximum inhibition of
33.64 (IC50 = ND) at 0.5 mg/mL was observed with L2
followed by 21.69 % (IC50 = 0.0986417 nM), at
0.0005 mg/mL with the compound L1. It is also interesting
to note that the maximum percentage inhibitions for all the
compounds were observed for HeLa cell line. Maximum
inhibitions were 35.0 % (IC50 = 20.35 nM, 0.05 mg/mL;
L1) followed by 28.57 % (IC50 = ND, 0.5 mg/mL, C1). On
the other hand, compound L2 showed 53.15 % inhibition
with IC50 = ND, 0.5 mg/mL followed by 39.55 % inhibi-
tion of C2 with IC50 = 0.04172 nM at 0.0005 mg/mL
concentration. All the four tested compounds revealed
better inhibition against HeLa cells in the order
L2 [ C2 [ C1 [ L1. These results indicated quite good
anti-cancer activities for the reported compounds.
Conclusion
The results presented in this article demonstrate good
yields of Knoevenagel condensates of curcumin I using
piperidine as a catalyst. The structure of intermediate
products, ligands and metal complexes were established by
chromatographic, spectroscopic, and other techniques. The
synthesized ruthenium metal ion complexes were of octa-
hedral geometries. The hypochromicities and batho-
chromicities indicated strong binding of these complexes
with DNA. Fluorescence intensity shifts of the complex–
DNA adduct also showed good binding of these com-
pounds with DNA. Cytotoxicities of the compounds were
studied on human RBCs, and it was observed that the
reported compounds were significantly less toxic than the
123

Med Chem Res (2013) 22:1386–1398
1397
Ferrari E, Lazzari S, Marverti G, Pignedoli F, Spagnolo F, Saladini M
(2009) Synthesis, cytotoxic and combined cDDP activity of new
stable curcumin derivatives. Bioorg Med Chem 17:3043–3052
Frasca D, Ciampa J, Emerson J, Umans RS, Clarke MJ (1996) Effects
of hypoxia and transferrin on toxicity and DNA binding of
ruthenium antitumor agents in HeLa cells. Metal Based Drugs
4:197–209
John VD, Kuttan G, Krishnankutty K (2002) Anti-tumor studies of
metal chelates of synthetic curcuminoids. J Exp Clin Cancer Res
21:219–224
Kelloff GJ, Crowell JA, Hawk ET, Steele VE, Lubet RA, Boone CW,
Covey JM, Doody LA, Omenn GS, Greenwald P, Hong WK,
Parkinso DR, Bagheri D, Baxter GT, Blunden M, Doeltz MK,
Eisenhauer KM, Johnson K, Knapp GG, Longfellow DG,
Malone WF, Nayfield SG, Seifried HE, Swall LM, Sigman CC
(1996) Clinical development plans for cancer chemopreventive
agents. J Cell Biochem 26:72–315
standard anti-cancer drug letrazole. The initial in vitro
screening showed that the reported compounds, i.e., L1, L2,
C1, and C2 possessed relatively high cytotoxicities against
HeLa cells, with moderate activities against HepG2, MDA-
MB-231 and HT-29 cells. IC50 values were in the range of
0.004 to 20.35 nM for different cell lines. Briefly, the
reported compounds may have potential as anti-cancer
medication in the future, particularly for the treatment of
cervical cancer.
Acknowledgments The authors are thankful to University Grants
Commission (UGC), New Delhi for providing UGC-BSR research
fellowship to A. H.
Li QS, Yang P, Wang HF, Guo ML (1996) Diorganotin (IV)
antitumor agent. (C2H5)2SnCl2 (phen)/nucleotides aqueous and
solid-state coordination chemistry and its DNA binding studies. J
Inorg Biochem 64:181–195
Lina L, Lee KH (2006) Structure–activity relationships of curcumin
and its analogs with different biological activities. Stud Nat Prod
Chem 33:785–812
Liu J, Zhang H, Chen C, Deng H, Lu T, Ji L (2003) Interaction of
macrocyclic copper (II) complexes with calf thymus DNA:
effects of the side chains of the ligands on the DNA-binding
behaviors. Dalton Trans 1:114–119
Lo Tempio MM, Veena MS, Steele HL, Ramamurthy B, Ramalingam
TS, Cohen AN, Chakrabarti R, Srivatsan ES, Wang MB (2005)
Curcumin suppresses growth of head and neck squamous cell
carcinoma. Clin Can Res 11:6994–7002
Manoharan S (1996) Inhibition of 4-nitroquinoline-l-oxide-induced
oral carcinogenesis by plant product. J Clin Biochem Nutr 21:
141–149
Mestroni G, Alessio E, Sava G, Pacor S, Coluccia M, Boccarelli A
(1993) Water soluble ruthenium(III)-dimethyl sulfoxide com-
plexes: chemical behavior and pharmaceutical properties. Met
Based Drugs 1:41–63
Midorikawa K, Kawanishi S (2001) Superoxide dismutases enhance
H2O2-induced DNA damage and alter its site specificity. FEBS
Lett 495:187–190
Modukuru NK, Snow KJ, Perrin BS Jr, Thota J, Kumar CV (2005)
Contributions of a long side chain to the binding affinity of an
anthracene derivative to DNA. J Phys Chem B 109:11810–11818
Ohtsu H, Xiao Z, Ishida J, Nagai M, Wang HK, Itokawa H, Su CY,
Shih C, Chiang T, Chang E, Lee Y, Tsai MY, Chang C, Lee KH
(2002) Curcumin analogues as novel androgen receptor antag-
onists with potential as anti-prostate cancer agents. J Med Chem
45:5037–5042
Padhye S, Yang H, Jamadar A, Cui QC, Chavan D, Dominiak K,
McKinney J, Banerjee S, Dou QP, Sarkar FH (2009) New
difluoro Knoevenagel condensates of curcumin, their Schiff
bases and copper complexes as proteasome inhibitors and
apoptosis inducers in cancer cells. Pharma Res 26:1874–1880
Parka HC, Hahma ER, Parkb S, Kima HK, Yang CH (2005) The
inhibitory mechanism of curcumin and its derivative against b-
catenin/Tcf signaling. FEBS Lett 579:2965–2971
References
http://www.cancer.gov/clinicaltrials/search/view?cdrid=648267&
version=HealthProfessional&protocolsearchid=6994018. Acces-
sed 13 Feb 2012
Adams BK, Cai J, Armstrong J, Herold M, Lu YJ, Sun A, Snyder JP,
Liotta DC, Jones DP, Shoji M (2005) EF24, a novel synthetic
curcumin analog, induces apoptosis in cancer cells via a redox-
dependent mechanism. Anticancer Drugs 16:63–275
Aggarwal BB, Kumar A, Bharti AC (2003) Anticancer potential of
curcumin: preclinical and clinical studies. Anticancer Res
23:363–398
Ali I, Rahisuddin SK, Haque A, El-Azzouny A (2010) Natural
products: human friendly anticancer medications. Egypt Pharma
J 9:133–179
Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB (2007)
Bioavailability of curcumin: problems and promises. Mol Pharm
4:807–818
Andrushchenko VV, Leonenko Z, van de Sande H, Wieser H (2002)
Vibrational CD (VCD) and atomic force microscopy (AFM)
study of DNA interactions with Cr3?: VCD and AFM evidence
of DNA condensation. Biopolymers 61:243–260
Arjmand F, Jamsheera A (2011) DNA binding studies of new valine
derived chiral complexes of tin (IV) and zirconium (IV).
Spectrochim Acta A 78:45–51
Barton JK, Danishefsky AT, Goldberg JM (1984) Tris (phenanthro-
line) ruthenium (II): stereoselectivity in binding to DNA. J Am
Chem Soc 106:2172–2176
Brabec V, Novakova O (2006) DNA binding mode of ruthenium
complexes and relationship to tumor cell toxicity. Drug Resist
Updat 9:111–122
Chittilapilly PS, Yusuff KKM (2008) Synthesis, characterization and
biological properties of ruthenium (III) schiff base complex
derived from 3-hydroxyquionoxaline-2-carboxaldehyde and sal-
icylaldehyde. Ind J Chem 47A:848–853
Christie MS, Kenneth LR, David BW (2007) Assessing toxicity of
fine and nanoparticles: comparing in vitro measurements to in
vivo pulmonary toxicity profiles. Toxicol Sci 97:163–180
Coluccia M, Sava G, Loseto F, Nassi A, Boccarelli A, Giordano D,
Alessio E, Mestroni
G (1993) Antileukemic action of
Priya NP, Arunachalam S, Manimaran A, Muthupriya D, Jay-
abalakrishna C (2009) Mononuclear Ru(III) Schiff base com-
plexes: synthesis, spectral, redox, catalytic and biological
activity studies. Spectrochim Acta A 72:670–676
Qiu X, Liu Z, Shao WY, Liu X, Jing DP, Yu YJ, An LK, Huang SL,
Bu XZ, Huang ZS, Gu LQ (2008) Synthesis and evaluation of
curcumin analogues as potential thioredoxin reductase inhibitors.
Bioorg Med Chem 16:8035–8041
RuCl2(DMSO)4 isomers and prevention of brain involvement
on P388 leukemia and on P388/DDP subline. Eur J Can 29A:
1873–1879
Duvoix A, Blasius R, Delhalle S, Schnekenburger M, Morceau F,
Henry E, Dicato M, Diederich M (2005) Chemopreventive and
therapeutic effects of curcumin. Cancer Lett 223:181–190
Erkkila KE, Odom DT, Barton JK (1999) Recognition and reaction of
metallointercalators with DNA. Chem Rev 99:2777–2795
123

1398
Med Chem Res (2013) 22:1386–1398
Situ H, Bobek LA (2000) In vitro assessment of antifungal therapeutic
potential of salivary histatin-5, two variants of histatin-5, and
salivary mucin (MUC7) domain1. Antimicrob Agents Chemo-
ther 44:1485–1493
Xu ZD, Liu H, Xiao SL, Yang M, Bu XH (2002) Synthesis, crystal
structure, antitumor activity and DNA-binding study on the
Mn(II) complex of 2H–5-hydroxy-1,2,5-oxadiazo[3,4-f]1,10-
phenanthroline. J Inorg Biochem 90:79–84
Stephanos JJ, Farina SA, Addison AW (1996) Iron ligand recognition by
monomeric hemoglobin’s. Biochim Biophy Acta 1295:209–221
Wang Y, Yan ZY (2005) Synthesis, characterization and DNA-
binding properties of three 3d transition metal complexes of the
Schiff base derived from diethenetriamine with PMBP. Trans
Metal Chem 30:902–906
Wu JZ, Yuana L, Wu JF (2005) Synthesis and DNA binding of
l-[2,9-bis(2-imidazo[4,5-f] [1,10]phenanthroline)-1,10-phenan-
throline]bis[1,10-phenanthrolinecopper(II)]. J Inorg Biochem
99:2211–2216
Cell Titer 96 Non-Radioactive Cell Proliferation Assay. Technical
Bulletin #TB112, Promega Corporation USA. Revised 12/99
Zeller WJ, Fruhauf S, Chen G, Keppler BK, Frei E, Kaufmann M
(1991) Chemoresistance in rat ovarian tumours. Eur J Can
27:62–67
Zhang Q, Fu Y, Wang HW, Gong T, Qin Y, Zhang ZR (2008)
Synthesis and cytotoxic activity of novel curcumin analogues.
Chin Chem Lett 19:281–285
123